简单节杆菌17α-羟基-16β-甲基-孕甾-4,9(11)二烯-3,20-二酮脱氢酶产酶条件研究

目的优化简单节杆菌17α-羟基-16β-甲基孕甾-4,9(11)-二烯-3,20-二酮(简称HMPDD)脱氢酶产酶条件。方法采用摇瓶培养方法,考察培养基组成及发酵条件对HMPDD转化率的影响。结果较佳产酶培养基为:葡萄糖0.4%、玉米浆1.2%、KH2PO40.2%、蛋白胨0.3%。较佳产酶条件为:培养基初始pH7.0;接种量20%;摇瓶装量100mL/250mL三角瓶;接种后4h内添加0.05g/L底物作为诱导物对产酶有利;添加10-4mol/LCo2+和Mg2+,可使酶活分别较对照提高12.9%和6.1%;菌体培养12h时酶活达最大值,为24.46μmol/g·min。结论通过培养基组成及产酶条件的优化,酶活力有较大幅度的提高。     

简单节杆菌17α-羟基-16β-甲基-孕甾-4,9(11)二烯-3,20-二酮脱氢酶产酶条件研究

目的优化简单节杆菌17α-羟基-16β-甲基孕甾-4,9(11)-二烯-3,20-二酮(简称HMPDD)脱氢酶产酶条件。方法采用摇瓶培养方法,考察培养基组成及发酵条件对HMPDD转化率的影响。结果较佳产酶培养基为:葡萄糖0.4%、玉米浆1.2%、KH2PO40.2%、蛋白胨0.3%。较佳产酶条件为:培养基初始Ph7.0;接种量20%;摇瓶装量100mL/250mL三角瓶;接种后4h内添加0.05g/L底物作为诱导物对产酶有利;添加10^-4mol/Lco²⁺和Mg²⁺,可使酶活分别较对照提高12.9%和6.1%;菌体培养12h时酶活达最大值,为24.46μmol/g·min。结论通过培养基组成及产酶条件的优化,酶活力有较大幅度的提高。     

海葵来源真菌 Cochliobolus lunatus 产大环内酯化合物LL-Z1640-2 的发酵优化

目的 对海葵来源真菌 Cochliobolus lunatus (TA26-46) 进行发酵优化以提高该菌产大环内酯化合物 LL-Z1640-2 的产量。方法 运用单因素试验设计和正交试验设计的方法,对菌株产 LL-Z1640-2 的碳源、氮源、初始 pH 值、发酵时间等发酵条件进行了优化。结果 得到了该菌株产 LL-Z1640-2 的优化发酵条件,其中,复合型培养基中可溶性淀粉 30.0 g.L–1,麦芽糖 20.0 g.L–1,硝酸钠 1.0 g.L–1,蛋白胨 3.0 g.L–1;初始 pH 6.0,培养时间 84 h。结论 在优化发酵条件下,LL-Z1640-2 产量可达到 (119.8±1.6) mg.L–1,比优化前提高了 3.6 倍。     

重组人抗HBsAg单链抗体工程菌的中试发酵工艺研究

目的研究表达重组人抗HBsAg单链抗体工程菌的中试发酵工艺。方法首先采用摇瓶培养,对培养基配方、pH值、诱导表达时机和诱导剂量进行优化,确定基本发酵参数,然后在30L发酵罐上进行中试规模发酵培养。结果在摇瓶培养时,发现含有甘油和微量元素等成分的改良培养基效果明显优于LB培养基和2×YT培养基,另外,工程茵的最佳pH值为7.0,最佳诱导时机为对数中期,最佳诱导剂浓度为0.4mmoL/L异丙基-1-硫代-β-呋喃半乳糖,最佳诱导时间为4h,将摇瓶培养确定的关键参数应用到30L发酵罐中,控制溶氧大于30%,进行3批发酵试验,发现发酵产物的湿菌产量可达58-61.9g/L,目的蛋白质表达量可达28.9%-31.2%以上。结论建立了工程菌M15[pQE-scFv]的中试发酵工艺,为进一步开发打下良好基础。     

产腺苷甲硫氨酸酿酒酵母半胱氨酸补料工艺的研究

对酿酒酵母生物合成腺苷甲硫氨酸(S-adenosylmethionine,SAM)发酵过程进行工艺优化,研究了半胱氨酸添加量、半胱氨酸添加时间、碳源对菌体浓度和SAM产量的影响。结果表明,在10 L发酵罐中,发酵16 h时,补加半胱氨酸至浓度2 mmol/L,发酵36 h时菌体(DCW)和SAM浓度分别达到15.40 g/L和4.11 g/L;在此基础上,更换糖蜜为碳源,并在还原糖浓度低于5 g/L时,流加糖蜜,相当于还原糖的流加速率为0.8 g/L.h,发酵36 h时菌体浓度和SAM产量分别为15.50 g/L和5.02 g/L;经过发酵工艺优化,发酵液中SAM浓度提高了43.8%。     

β-环糊精包合技术在微生物转化生产氢化可的松中的应用

为提高甾体生物转化的底物投料浓度，采用超声法制备底物RS—β—环糊精包合物。以新月弯孢霉为实验菌种，培养后加入溶有包合物的磷酸盐缓冲液制备氢化可的松。结果表明，采用β-环糊精包合后甾体底物浓度可由2g/L提高至3g／L，2L发酵罐中氢化可的松收率为57．8％。     

北极放线菌Streptomyces sp. MLA-21发酵条件优化

对北极放线菌Streptomyces sp.MLA-21的发酵条件进行优化,提高发酵液中抗肿瘤活性部位的产量。以发酵液对肺癌A549细胞的抑制率（IR）作为观测指标,利用响应面分析法对通过单因素实验和Plackett-Burman设计筛选出培养基初始pH值、黄豆浸出物含量,可溶性淀粉含量等关键因素进行优化。建立二次回归模型。得到菌株MLA-21发酵的最优条件为KNO₃1.0 g/L,可溶性淀粉23.04g/L,黄豆浸出物7.1g/L,K₂HPO₄ 0.5g/L,MgSO₄ 0.5g/L,NaCl 0.5g/L,pH 7.67,接种量:7.5%,培养温度:28℃,摇床转速:150 r/min。对优化结果进行验证发现优化后的培养基能够显著提高发酵液对A549细胞的抑制率和活性部位的产量。     

欧文氏菌发酵生产紫杉醇工艺的优化

考察了植物内生细菌欧文氏菌(Erwinia taxi)生产紫杉醇的摇瓶发酵和30 L罐发酵工艺条件。结果显示,培养基中添加不同的前体对紫杉醇产量影响显著。较高浓度的乙酸钠(>0.01 mmol/L)和较低浓度的苯丙氨酸(<0.05 mmol/L)有利于紫杉醇积累。在此基础上进行了30 L罐发酵试验,得优化培养基配方(g/L):大豆蛋白胨20,工业酵母抽提物5,氯化钠0.5,氯化钾0.1,葡萄糖4,氯化镁1,乙酸钠0.1,苯丙氨酸5×10-4;pH 6.5。发酵培养条件:接种量5%,发酵温度22℃,通气速率0.17 vvm,转速50 r/min,发酵时间60 h。在此条件下,30 L发酵罐中紫杉醇的产量为102 g/L。     

十二碳二元酸菌种发酵工艺优化

目的 以热带假丝酵母菌为试验菌株,通过发酵工艺优化,提高菌种发酵产酸量。方法 对发酵培养基中碳源和氮源等成分进行优化,同时研究了种龄、发酵培养温度、摇瓶转速、发酵接种量、发酵瓶装量和发酵pH值对发酵产酸量的影响。结果 发酵培养最适碳源为2 g/L蔗糖,最适氮源为3 g/L酵母浸膏,菌体发酵在28℃,200 r/min,25 mL/250 mL装量,以15%接种量条件下培养,发酵pH值调至7.0～7.5时,十二碳二元酸平均产量可达到130 g/L。结论 对发酵培养基碳氮源成分进行优化、发酵工艺进行优化,发酵产酸量提高至130 g/L。同时发酵培养基中添加氯化钠和硫酸镁后,均不利于菌种产酸。     

用根霉80322进行16α,17α-环氧-△~4-孕甾烯-3,20-二酮11α羟基化的副产物

11α-羟基-16α、17α-环氧-△~4-孕甾烯-3,20-二酮(Ⅱ)是合成可的松、强的松、肤轻松等甾体激素药物的重要中间体,由16α,17α-环氧-△~4-孕甾烯-3,20-二酮(Ⅰ)经霉菌氧化而得。上海工业微生物研究所从400余株野生梨的霉菌中,选出一株根霉80322新菌株,可在2％底物投料浓度,37℃发酵36～48h,使转化率达到55％以上,其主要发酵产物除11α-羟基-16     

The effects of phenytoin dosage on the induction of alpha 1-acid glycoprotein and antipyrine clearance in the dog

Doses of phenytoin from 90 to 800 mg/d were used to study induction of hepatic cytochrome P-450 and plasma alpha 1-acid glycoprotein in dogs. The antipyrine clearance was increased by 80%, which is equivalent to an increase in cytochrome P-450 of 140%, and the plasma glycoprotein concentration rose 200% at the highest dose of phenytoin used. Plasma concentrations of phenytoin were measured at each dose level to provide a definitive value for the amount of inducer present. These data were used to assess the concentration-response relationship for phenytoin inducing either cytochrome P-450 or the glycoprotein. A simple relationship between concentration and effect was not observed, suggesting a complex mechanism of induction.     

Induction of rat liver microsomal and nuclear cytochrome P-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.     

Induction of cytochrome P-450 isozymes upon repeated administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine administration at a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450IIA1, P-450IIB1, and P-450IIIA1, respectively. The induction of cytochrome P-450IIIB1 was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450IIB1/B2.     

The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats

The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.     

Expression of cytochrome P-450s and glutathione S-transferases in the rat liver during water deprivation: effects of glucose supplementation

Pharmacokinetic profiles of therapeutic agents change in dehydrated animals. The present study was designed to determine the expression of xenobiotic-metabolizing enzymes in the rat liver and the effect of glucose supplementation during water deprivation. Deprivation of water intake, which reduced food intake, resulted in no significant change in the cytochrome P-450 1A2, 2B1/2, 2C11 and 3A1/2 expression. Cytochrome P-450 2E1, however, was three-fold induced with an increase in the mRNA. Rehydration of 48-h water-deprived rats for the next 24 h with free access to foods restored the P-450 2E1 level to that of the control, although rehydration with 20% food supply failed to normalize the P-450 2E1 expression. Water deprivation caused a reduction in the plasma insulin level, which was prevented by rehydration with a sufficient food supply. The plasma insulin level was inversely related to the P-450 2E1 expression. Glucose feeding instead of foods during dehydration prevented P-450 2E1 induction in the absence of recovering the plasma insulin level. Western blot analysis revealed that the hepatic rGSTA2 level was 30% decreased in dehydrated rats, whereas the rGSTA3, M1 and M2 expression was not affected. Suppression of rGSTA2 accompanied a reduction in the mRNA. Glucose feeding further reduced rGSTA2 expression. The data indicated that expression of major P-450s and glutathione S-transferases, except P-450 2E1, was not greatly affected by water deprivation and that the P-450 2E1 induction and a decrease in plasma insulin resulted from the reduction in food intake but not from dehydration per se. Glucose supplementation restored P-450 2E1 expression but further suppressed rGSTA2 expression during water deprivation.     

Effect of 17 alpha-ethynylestradiol on the induction of cytochrome P-450 by 3-methylcholanthrene in cultured chick embryo hepatocytes

This study investigated the effects of estrogens on the induction of cytochrome P-450 by polycyclic aromatic hydrocarbons in primary cultures of chick embryo hepatocytes. Exposure to polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene led to 2- to 3-fold increases of cytochrome P-450. The amount of cytochrome P-450 induced by 3-methylcholanthrene was increased 40-50% when the synthetic estrogen, 17 alpha-ethynylestradiol, was also present. The rate of decay of cytochrome P-450 in the presence of cycloheximide as measured spectrophotometrically was similar in cells previously treated with either 3-methylcholanthrene or 3-methylcholanthrene plus 17 alpha-ethynylestradiol, suggesting that 17 alpha-ethynylestradiol did not affect the stability of the 3-methylcholanthrene-induced cytochrome P-450. In contrast, 17 alpha-ethynylestradiol did not potentiate the induction of cytochrome P-450 by phenobarbital-like inducers, such as 2-propyl-2-isopropylacetamide, as indicated by a lack of increase in both the content of cytochrome P-450 and benzphetamine demethylase activity. The naturally occurring estrogens, 17 beta-estradiol and estrone, and the synthetic estrogen, diethylstilbestrol, did not affect cytochrome P-450 induction by 3-methylcholanthrene, suggesting that the effect of 17 alpha-ethynylestradiol was not mediated via the estrogen receptor. We investigated whether the amount of cytochrome P-450 increased in the presence of 17 alpha-ethynylestradiol was the same or different from that induced by 3-methylcholanthrene. Treatment with 17 alpha-ethynylestradiol alone resulted in a small increase in ethoxyresorufin deethylase activity. The enzymatic activities of 7-ethoxyresorufin and aryl hydrocarbon hydroxylase, when expressed per cytochrome P-450 content, were identical in microsomes from cells treated with either 3-methylcholanthrene or the combination of 3-methylcholanthrene and 17 alpha-ethynylestradiol. The data suggest that the additional cytochrome P-450 induced by the combination of 17 alpha-ethynylestradiol and 3-methylcholanthrene was the same isozyme as that induced by 3-methylcholanthrene alone.     

Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA

1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3 (2100 mg/m3). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%) and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in vivo in both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.     

The effect of age on inducibility of various types of rat liver cytochrome P-450.

1. The content and specific activities of inducible cytochrome P-450 enzymes were determined in liver microsomes of rats of various ages after maximal induction with phenobarbital, isosafrole of 3-methylcholanthrene, and in untreated animals. 2. With age an increase in liver weight was observed both in untreated rats and in maximally induced ones; the microsomal protein content/g of liver decreased with age in untreated animals but not in induced ones. Total cytochrome P-450 content/mg microsomal protein remained unchanged with age in all experimental groups. 3. Immunologically detectable levels of cytochrome P4501A1/1A2 and 2B1/2B2 remain unchanged with age both in untreated animals and in maximally induced ones. 4. Several cytochrome P-450 activities showed an age-related decrease in untreated animals, but no change with age was observed in the activities of cytochrome P4501A1, 2A2 and 2B1/2B2 in rat liver microsomes. This indicates that ageing affects only the activity of some constitutive forms of cytochrome P-450 in male rats, but not the activity of inducible types of P-450. 5. Although previous results indicated decreased inducibility of the cytochrome P-450 mRNA levels with age, the present study clearly demonstrates that this is not reflected in decreased enzyme levels or activities after maximal induction. From this it is concluded that the decreased mRNA levels might rather be reflected in a decreased rate at which maximal induction can be achieved.     

Ontogeny of the chicken cytochrome P-450 enzyme system. Expression and development of responsiveness to phenobarbital induction

The sensitivity of the developing embryo to toxins and drugs is highly dependent on the state of development of the cytochrome P-450 system. Previous work in this laboratory has demonstrated the genotoxicity of aflatoxin B1 (AFB1) to the chicken embryo at 3 days of incubation (DI) and induction of AFB1 genotoxicity by phenobarbital at 7 DI. In this study, the basal and 24-hr phenobarbital (PB) induced levels of aminopyrine-N-demethylase (AMPD) and cytochrome P-450 were assayed in hepatic microsomes from 7 DI to 36 days posthatching (PH) and in microsomes from whole embryos at 5 DI. A dose-response for induction by PB was observed in embryonic hepatic microsomes as early as 7 DI, whereas a low level of cytochrome P-450 was detected in control 7 DI microsomes using the reduced CO vs oxidized CO difference spectrum. Basal levels of AMPD and cytochrome P-450 in hepatic microsomes increased steadily throughout development as did the responsiveness of the embryonic liver to induction with PB. Hepatic microsomes from control and PB-induced chickens had the highest AMPD activities posthatching particularly from 1 to 3 days PH. Maximal induced levels, which were 2- to 3-fold over control throughout development, ranged from 1.22 at 7 DI to 12.72 nmol HCHO/mg protein/min at 2 days PH. The potency of PB as an inducer increased about 1000-fold between 7 DI and hatching. PB induction did not increase the specific activity of AMPD at any period of development. The specific activity of AMPD posthatching increased about 3-fold above embryonic levels, indicating the development of a cytochrome P-450 complex more active toward aminopyrine in the neonatal period.     

Developmental regulation of cytochrome P-450 genes in the rat

Synthetic oligomer probes were used in hybridization experiments to investigate the developmental regulation of cytochrome P-450 (P-450) genes in rat liver. Transplacental induction by phenobarbital of P-450b and P-450e mRNAs was not detectable in fetal rat livers prior to day 21 of gestation. The levels of these mRNAs increased approximately 2-fold from gestational day 21 to day 22 in phenobarbital-induced liver. P-450b and P-450e mRNAs were shown to be adenylated and the fractions associated with polysomes were similar in both fetal and adult livers. No P-450b or P-450e mRNAs were detected in fetal lung and kidney RNA preparations regardless of pretreatment. Southern blot data utilizing fetal liver DNA suggests that responsiveness to xenobiotic induction during development is not attained by rearrangement of P-450b or P-450e genes. Experiments with probes specific for P-450c and P-450d failed to detect their respective mRNAs in fetal livers from 3-methylcholanthrene (3-MC)-treated or untreated rats. Both species were detectable in 3-MC-treated rats 1 week after birth. The levels of 3-MC-inducible P-450c and P-450d mRNAs increased with age and peaked approximately 3 weeks after birth. Hepatic P-450d mRNA levels in 3-MC-treated or control rats was consistently higher than P-450c mRNA levels at all ages studied.